The present disclosure relates to a minute particle analyzing device and method, and more particularly to a minute particle analyzing device for optically analyzing the characteristics of minute particles such as cells and microbeads.
One minute particle analyzing device applies light to minute particles flowing in a channel formed in a flow cell or on a microchip and detects scattered light from each minute particle or fluorescence generated from each minute particle itself or from a fluorescent material labeled on each minute particle, thus measures the optical characteristics of each minute particle. The minute particle analyzing device further performs sorting of a population from the minute particles, wherein the population is determined to satisfy predetermined conditions from the result of the measurement of the optical characteristics. In particular, such a device for measuring the optical characteristics of cells as the minute particles or sorting a population of cells satisfying predetermined conditions is called a flow cytometer or cell sorter.
For example, Japanese Patent Laid-open No. 2007-46947 (hereinafter, Patent Document 1) discloses a flow cytometer comprising a plurality of light sources for emitting a plurality of exciting light beams having different wavelengths with a predetermined period and different phases and a light guide member for guiding the plurality of exciting light beams to a common incident optical path and condensing the resultant light beam along the common incident optical path to a stained particle. That is, this flow cytometer includes a plurality of light sources for emitting a plurality of exciting light beams having different wavelengths, a light guide member for guiding the plurality of exciting light beams to a common incident optical path and condensing the resultant light beam along the common incident optical path to a stained particle, and a plurality of fluorescence detectors for detecting fluorescence generated from the particle by the irradiation with the exciting light beams and outputting a fluorescence signal (see claims 1 and 3 and FIGS. 1 and 3 in Patent Document 1).
Such an existing minute particle analyzing device as disclosed in Patent Document 1 adopts a light applying path having a high optical magnification in order to enlarge the very small spot size of the light beam emitted from each light source so that the spot size of the light beam applied to a sample flow of minute particles becomes sufficiently larger than the width of the sample flow.
FIG. 9 is a schematic diagram showing a light applying path and a light detecting path in an existing minute particle analyzing device. Referring to FIG. 9, light beams (exciting light) emitted from a plurality of light sources 111 are respectively collimated by a plurality of collimator lenses 112, and the resultant parallel light beams from the collimator lenses 112 are respectively reflected on a plurality of mirrors 113 to propagate along a common optical axis. The resultant light beam propagating along this common optical axis is condensed by a condenser lens 114 to irradiate each minute particle P in a sample flow S flowing in a channel formed in a flow cell or on a microchip. In FIG. 9, the arrow F denotes a flowing direction of the sample flow S and a sheath flow in the flow cell.
By the irradiation with the exciting light, fluorescence is generated from each minute particle P or a fluorescent material labeled on each minute particle P. The fluorescence thus generated is collimated by an objective lens 121 and next sequentially passed through a plurality of wavelength filters 122. At this time, a predetermined wavelength region of the fluorescence is separated by each wavelength filter 122 and next detected by a detector 123 provided for each wavelength filter 122. In each detector 123, the detected fluorescence is converted into an electrical signal.
The required spot size of the light beam on the sample flow S is about 10 to 100 μm, for example, normally about 20 μm. In contrast, the spot size of the light beam emitted from each light source 111 is about 0.5 to 2.0 μm, for example, normally about 1 μm. In this case, the optical magnification determined by the ratio in focal length between the condenser lens 114 and each collimator lens 112 in the light applying path is set to about 20.
In the light applying path having such a high optical magnification, a deviation of 1 μm in relative position between each light source 111 and the corresponding collimator lens 112, for example, causes a deviation of 20 μm in spot position of the light beam on the sample flow S. This deviation in spot position is the same value as that of the spot size of the light beam on the sample flow S. That is, the light beam from each light source 111 cannot be applied to each minute particle P in the sample flow S, so that a detection signal cannot be obtained.
FIG. 10B is a graph schematically showing the distribution of light intensity of the light spot on the sample flow S in the existing minute particle analyzing device. As shown in FIG. 10A, the light beam L condensed by the condenser lens 114 (see FIG. 9) is applied to each minute particle P in the sample flow S flowing in the channel formed in a flow cell or on a microchip. In this case, the distribution of light intensity of the light spot on the sample flow S becomes a Gaussian distribution. That is, the light intensity of the light spot on the sample flow S is large at the center of the light spot and weak in the periphery of the light spot. As a result, the deviation in position of the light spot on the sample flow S causes a large reduction in effective intensity of the light beam applied to each minute particle P, resulting in attenuation of the detection signal.
Japanese Patent Laid-open No. 2004-184217 (hereinafter, Patent Document 2) discloses a flow cytometer whose light applying path includes an optical fiber for applying laser light emitted from a laser oscillator to a sheath flow (see FIG. 1 and paragraph 0013 in Patent Document 2). This optical fiber is located between the laser oscillator and a beam expander, and functions to merely guide the laser light emitted from the laser oscillator to the beam expander. In patent document 2, there is no description about changing of the spot size of a light beam emitted from a light source by the use of an optical fiber.
In the existing minute particle analyzing device as shown in FIG. 9, the light applying path has a high optical magnification, so that when minute deviation occurs in relative position between a light source and a lens, for example, in the light applying path, the spot position of the light beam applied to the sample flow is largely deviated. Further, the distribution of light intensity of the light spot on the sample flow S is a Gaussian distribution, so that the deviation in position of the light spot on the sample flow causes a large reduction in effective intensity of the light beam applied to each minute particle.
Such minute deviation in relative position between a light source and a lens in a light applying path is easily produced by vibrations applied to the device or temperature changes or may be naturally produced with the elapse of time. Accordingly, the existing minute particle analyzing device has a problem such that the detection signal largely changes due to the deviation in position of the light spot on the sample flow, causing a reduction in stability of the device performance and in measurement accuracy.
There is accordingly a need to provide a minute particle analyzing device and method which can suppress the deviation in position of the light spot on the sample flow due to minute deviation in relative position between a light source and a lens, for example, in a light applying path, so that the light can be applied to each minute particle with high accuracy, thus obtaining a stable measurement performance.